The nuclear lamina is required for proper development and nuclear shape distortion in tomato

Abstract

The nuclear lamina in plant cells is composed of plant-specific proteins, including nuclear matrix constituent proteins (NMCPs), which have been postulated to be functional analogs of lamin proteins that provide structural integrity to the organelle and help stabilize the three-dimensional organization of the genome. Using genomic editing, we generated alleles for the three genes encoding NMCPs in cultivated tomato (Solanum lycopersicum) to determine if the consequences of perturbing the nuclear lamina in this crop species were similar to or distinct from those observed in the model Arabidopsis thaliana. Loss of the sole NMCP2-class protein was lethal in tomato but is tolerated in Arabidopsis. Moreover, depletion of NMCP1-type nuclear lamina proteins leads to distinct developmental phenotypes in tomato, including leaf morphology defects and reduced root growth rate (in nmcp1b mutants), compared with cognate mutants in Arabidopsis. These findings suggest that the nuclear lamina interfaces with different developmental and signaling pathways in tomato compared with Arabidopsis. At the subcellular level, however, tomato nmcp mutants resembled their Arabidopsis counterparts in displaying smaller and more spherical nuclei in differentiated cells. This result argues that the plant nuclear lamina facilitates nuclear shape distortion in response to forces exerted on the organelle within the cell.

Keywords: Leaf morphology; nuclear lamina; nuclear shape; nuclear size; root growth; tomato.

Fig. 1.

Comparison of NMCP proteins in tomato and Arabidopsis. (A) A distance tree constructed using an alignment limited to the conserved coiled-coil domains of CRWN/NMCP proteins in Arabidopsis and tomato (bootstrap values out of 100). Arabidopsis CRWN1, 2, and 3 are members of the NMCP1 clade, along with their tomato homologs, NMCP1A and NMCP1B. CRWN4 in Arabidopsis and its tomato ortholog, NMCP2, partition into the NMCP2 clade. (B) Domain structure of NMCP proteins in tomato with reference of Arabidopsis NMCP1-type (CRWN1) and NMCP2-type (CRWN4) proteins. Numbers refer to amino acid coordinates. The size and position of coiled-coil regions are shown in black, while a conserved C-terminal motif is denoted in blue. The N-terminal region, which is missing in NMCP1A from tomato, is designated in yellow.

Fig. 2.

Growth characteristics of nmcpa1 and nmcp1b mutants. (A) Images of the shoots of a wild-type M82 individual, a representative nmcp1a-1 homozygote, and two sibling nmcp1b-2 homozygotes; all plants were grown in parallel and are 23 d old. Scale bar, 1 cm. (B) Shoot fresh weight of M82 wild-type individuals (n=9) compared with nmcp1a-1 homozygotes (n=8) and nmcp1b-2 homozygotes (n=9); all plants were grown in parallel and were 24 d old when shoot tissue was harvested. Samples that share a letter designation (a, b, c) were not statistically different based on one-way ANOVA with post-hoc Tukey HSD tests. (C) Main root growth rate of wild-type M82 (2.5 ± 0.3 cm d^–1, n=7), nmcp1a-2 homozygotes (2.6 ± 0.3 cm d^–1, n=6), and nmcp1b-2 homozygotes (0.92 ± 0.3 cm d^–1, n=8). Student’s t-test for each mutant compared with the wild type; ***P<0.001 for the wild type versus nmcp1b-2, while the nmcp1a-2 genotype was not significantly different from the wild type.

Fig. 3.

Mutations in the NMCP1 genes reduce nuclear size and alter nuclear shape in guard cells. We imaged DAPI-stained nuclei from guard cells on the abaxial (lower) surface of the terminal leaflet of the third leaf of 5- to 7-week-old plants of the M82 wild type and homozygotes for the indicated alleles of either NMCP1 paralog. (A and B) Box-and-whisker plots displaying nuclear size (A), as reflected by cross-sectional area, or nuclear shape (B), where the roundness index equals the inverse of the aspect ratio (long/short axis). The numbers of nuclei measured: wild type (n=71), nmcp1a-1 (n=81), nmcp1a-2 (n=81), nmcp1b-1 (n=74), and nmcp1b-2 (n=76). **P<0.01 based on a one-way ANOVA with post-hoc Tukey HSD test for each genotype in comparison with the wild-type M82 sample. (C) Representative DAPI-stained guard cell nuclei from the different genotypes (scale bar, 10 μm). Note that leaf guard cells in cultivated tomato are 2n (Jacobs and Yoder, 1989); therefore, endopolyploidy is not a complicating factor in this analysis.

Fig. 4.

Genetic behavior of nmcp1a and nmcp1b loss-of-function mutations. (A) Transmission of nmcp1a and nmcp1b mutations in a segregating F₂ population. (B) Segregation of an nmcp1b mutation in the absence of NMCP1A function after self-pollination of 1A^–/–1B^+/– parents. (C) Results of reciprocal crosses between wild-type individuals and 1A^–/–1B^+/– individuals to examine the co-transmission of the nmcp1a and nmcp1b alleles through either the male or female gametophyte. ns, not significant.

Fig. 5.

Developmental defects in seedlings carrying a single NMCP1A allele in the absence of NMCP1B function. (A) Crossing scheme to recover 1A^+/–1B^–/– seeds, which show no or delayed germination. (B) Morphology of a 1A^+/–1B^–/– seedling (14 d old) dissected from a late germinating seed and subsequently grown on solid agar medium supplemented with sucrose; note abnormal cotyledon development, the swollen green primary root axis, and delayed rootlet growth.

Fig. 6.

Alteration in the N-terminal domain of NMCP2 reduces nuclear size and changes the nuclear shape in guard cells. We imaged DAPI-stained nuclei from guard cells on the abaxial (lower) surface of the terminal leaflet of developmentally matched leaves from 3-week-old plants of the M82 wild type and nmcp2-3 homozygotes. (A and B) Box-and-whisker plots displaying nuclear size (A), as reflected by cross-sectional area, or nuclear shape (B), where the roundness index equals the inverse of the aspect ratio (long/short axis). The number of nuclei measured is 186 for each sample, which was derived from two biological replicates. **P<0.01 based on a one-way ANOVA with post-hoc Tukey HSD test.